Project Summary Pancreatic cancer (PC) is one of the most devastating disease with very low survival rates. PC is characterized by the presence of a very dense desmoplastic stroma, which hinders the delivery of drugs to the tumor's core. Recent evidence suggests that the Receptor for Advanced Glycation End products (RAGE) contributes significantly to the progression of PC and to the formation of desmoplasia. RAGE is a cell-surface receptor that is activated by extracellular ligands. High levels of RAGE ligands have also been found in abundance in PC tumor stroma. We have identified anti-RAGE antibodies that reduced significantly the in vitro proliferation of PC cells. Given this, the goal of this proposal is to demonstrate that anti-RAGE antibodies can reduce PC tumor growth by both decreasing PC cell proliferation and tumor desmoplasia. We will test our hypothesis through four Specific Aims. In Aim 1, we will measure the binding affinity of new anti-RAGE antibodies for RAGE, and identify the binding epitopes of these anti-RAGE antibodies. These antibodies will be generated from hybridomas. Measurements will be performed in vitro, by ELISA and surface plasmon resonance. The epitopes of the antibodies will be determined by using peptide arrays of RAGE. In this aim, we will also measure the binding of the antibodies to cell surface RAGE by flow cytometry and fluorescence microscopy. In Aim 2, we will test whether the anti-RAGE antibodies can inhibit the proliferation, migration, or invasion of PC cells. We will also determine the molecular mechanisms of RAGE inhibition by anti-RAGE antibodies. The levels of key signaling molecules that are activated in PC tumors will be determined by Western blot analysis and compared after treatment with the antibodies. In Aim 3, we will test the abilities of anti-RAGE antibodies to inhibit fibroblasts' activation in 3D-spheroids. These spheroids will be generated from co-cultures of PC cells with fibroblasts. The mechanisms of inhibition by the antibodies will be studied by investigating changes in the levels of extracellular matrix proteins and chemokines secreted by activated fibroblasts. In Aim 4, we will test the efficacy of our antibodies in the KPC mouse model of PC. KPC mice develop spontaneously pancreatic tumors that resemble human ones. We will compare the growth of PC tumors in animal treated and non-treated with anti-RAGE antibodies. We will also investigate whether treating the tumor-bearing mice with anti-RAGE antibodies results in decreased desmoplasia and in increased sensitivity toward gemcitabine. Our proposed research will provide a better understanding of the molecular mechanisms of PC development and of the role of RAGE in PC. Our project could also be translated into a new therapeutic strategy to treat PC patients.